Food safety related to contamination of agricultural products by pathogenic fungi is an important concern for the agricultural community. Indeed, pathogenic microbes can contaminate agricultural crops and products and produce acutely poisonous, teratogenic or carcinogenic toxins. Because these toxins pose a grave threat to human, and animal, health, the detection and control of pathogenic fungi and the toxins they produce is imperative.
Particularly egregious pathogenic fungi are the aflatoxigenic aspergilli, Aspergillus flavus and Aspergillus parasiticus. These fungi are ubiquitous on crop plants important to agriculture, and under favorable conditions of temperature and humidity, are known to produce aflatoxins, which are among the most toxic and carcinogenic substances known to humankind. Among the many ill effects of aflatoxins are acute necrosis, cirrhosis, and carcinoma of the liver. Furthermore, the consumption of large amounts of aflatoxins over a short period of time can result in acute and deadly aflatoxosis. Thus, aflatoxin contamination is a significant food safety issue.
Because aflatoxins are extremely toxic and carcinogenic, strict threshold levels have been set for aflatoxin levels in human consumed food e.g., tree nuts, e.g., almonds, filberts, etc. Thus, in the U. S., the Food Drug Administration (FDA) has set threshold levels for aflatoxins in human consumed food. For example, the FDA has set a level of at 20 parts per billion (ppb) total aflatoxin content for ready to eat almonds, and even more stringent limits have been set in other countries e.g., countries of the European Union.
Aflatoxins are typically detected directly using, for example, thin layer chromatography (TLC), TLC in conjunction with mass spectrometry (see e.g., W. F. Haddon, et al. (1971) Anal. Chem., 43 (2):268-270), HPLC, radioimmunoassay, and enzyme linked immunosorbent assay. The presence of aflatoxins may also be inferred from the from volatile emissions indicative of aflatoxigenic fungi (see e.g., Jelen, H. H.; and Grabarkiewicz-Szczesna, J. (2005) J. Agric. Food Chem. 53:1678-1683; Wright, M. S. et al. (2000) Toxicon 38:1215-1223; Schnurer, J. et al., (1999) Fungal Gen. Biol. 27:209-217; Zeringue, H. J.; et al. (1993) Appl. Environ. Microbiol. 59:2264-2270).
Unfortunately, currently used methods are time consuming and rely on the isolation of the aflatoxin or the isolation and cultivation of the aflatoxigenc organism prior to implementation of the analysis (see e.g., Sunesson, A.-L.; et al. (1995) Appl. Environ. Microbiol. 61:2911-2918; Scotter, J. M.; et al., (2005) J. Microbiol. Methods 63:127-134). Thus, current methods cannot detect aflatoxin contamination early e.g., post harvest when contamination levels are low, and contamination has not spread throughout the entire harvested batch.
Because of the extreme toxicity of aflatoxins, the ubiquity of aflatoxigenic aspergilli on important agricultural crops, the consequences to both human and animal health from aflatoxin ingestion, and the possibility of spread of aflatoxigenic fungi and their associated aflatoxins from a “hot spot” of contamination to an entire batch of agricultural crop, early detection and field detection are imperative. Thus, what is needed in the art, are methods capable of early detection, which can be deployed for rapid, reliable detection under field conditions where the aflatoxin and aflatoxigenic fungi are co-existent with other fungal species in their natural fungal bouquet.
Fortunately, as will be clear from the following disclosure, the present invention provides for these and other needs.